A coiled-coil-based design strategy for the thermostabilization of G-protein-coupled receptors

Structure elucidation of inactive-state GPCRs still mostly relies on X-ray crystallography. The major goal of our work was to create a new GPCR tool that would provide receptor stability and additional soluble surface for crystallization. Towards this aim, we selected the two-stranded antiparallel coiled coil as a domain fold that satisfies both criteria. A selection of antiparallel coiled coils was used for structure-guided substitution of intracellular loop 3 of the β3 adrenergic receptor. Unexpectedly, only the two GPCR variants containing thermostable coiled coils were expressed. We showed that one GPCR chimera is stable upon purification in detergent, retains ligand-binding properties, and can be crystallized. However, the quality of the crystals was not suitable for structure determination. By using two other examples, 5HTR2C and α2BAR, we demonstrate that our approach is generally suitable for the stabilization of GPCRs. To provide additional surface for promoting crystal contacts, we replaced in a structure-based approach the loop connecting the antiparallel coiled coil by T4L. We found that the engineered GPCR is even more stable than the coiled-coil variant. Negative-staining TEM revealed a homogeneous distribution of particles, indicating that coiled-coil-T4L receptor variants might also be promising candidate proteins for structure elucidation by cryo-EM. Our approach should be of interest for applications that benefit from stable GPCRs.

. Design rationale of β3AR-coiled-coil chimeras. (a) Side view (left panel) of ribbon representation of crystal structure of β2-adrenergic receptor transmembrane helices 5 (green) and 6 (blue) that interact in an antiparallel coiled-coil like manner at the cytosolic side (PDB code: 2RH1) 19 . Interacting amino-acid residues are shown as sticks. Sequences of the interacting segments of transmembrane helices 5 and 6 are shown in the right panel. Residues at the hydrophobic heptad-repeat positions a, a′, d and d′ are shown in bold and numbered according to their position in the wild-type protein. N-and C-termini are indicated. (b) Example of chimeric β3AR. In-register heptad-repeat fusion of the antiparallel coiled coil of the Thermus thermophilus seryl-tRNA synthetase (SRS, cc3.1) (yellow) and the human β3-adrenergic receptor (blue). Introduction of three amino-acid residues at the junction (red) was necessary to obtain a continuous heptad-repeat pattern. Amino-acid residues at the e′ and f positions of the junction were chosen to introduce two additional attractive salt bridges into cc3. 1 and potentially further stabilize the protein. The heptad-repeat pattern is indicated. Potential salt bridges are indicated by arrows. (c) β3AR-cc3.1 model. β3AR is shown in blue, the cc3.1 coiled coil in yellow and residues at the junction in red. The model was generated by AlphaFold2 7 . (d) Western blot analysis demonstrating the expression of B3AR-cc3.1 (lane 1). The migration of marker proteins is shown (M).

Results and discussion
Design rationale of engineered GPCR variants. The design rationale of our GPCR variants is based on the observation that at the cytoplasmic site in the crystal structure of β2AR, transmembrane helices 5 and 6 are interacting over a short stretch of residues in a way that is very similar to an antiparallel coiled coil 19 (PDB code: 2RH1). Specifically, residues Phe223 and Ala226 of helix 5 that can be assigned the hydrophobic heptad-repeat positions a and d, respectively, interact with His269 and Leu272 at positions a′ and d′, respectively, of helix 6 ( Fig. 1a). β3AR was selected because it is the closest relative of β2AR, for which no high-resolution structure was available at the time. The idea behind the design was to replace ICL3 of β3AR by a series of antiparallel coiled coils and thereby extend the sequence corresponding to coiled-coil-like structure seen in β2AR (Fig. 1b,c). The coiled coil would therefore act like a clamp on β3AR transmembrane helices 5 and 6 that would hopefully stabilize the entire GPCR and provide additional soluble surface for crystallization. The coiled coils were inserted into ICL3 between Ala231 and Glu286 (Fig. 1b). Coiled-coil positions e, e′, g and g′ that flank the hydrophobic a, a′, d and d′ positions are frequently occupied by charged residues that form g to g′ and e to e′ type salt bridges that can further stabilize the structure 30 . To this end, amino-acid residues at the junction were chosen to optimize attractive electrostatic interactions between helices (Fig. 1b).
Towards this end, the RCSB Protein Data Bank, CC+ 31 and the literature was screened to identify suitable antiparallel coiled-coil candidates for substituting ICL3 of β3AR. The main criterion for selecting coiled coil fusion partners was that they form a stable two-stranded antiparallel structure alone. This applied to all the coiled coils except cc6, which, based on its extended structure, is likely to be stable alone. Ideally, a crystal structure would be available for the candidate coiled coils. Furthermore, we were also searching for coiled coils with different properties, including size, stability, self-interaction or additional structures. The heptad-repeat register was identified from the papers and the PDB files describing the structures and compared to coiled-coil prediction algorithms and analysis software.
Based on their characteristics, six antiparallel coiled coils were selected (Supplementary Fig. S1 and Supplementary Table S1). cc1 is derived from a bacteriophage serine integrase that plays a key role in the integration of the viral genome through self-interactions between the coiled-coil domains 32 . Based on its small size (three heptad repeats), the coiled coil is not expected to affect G-protein binding of the β3AR chimera. cc2 originates from a viral nucleocapsid protein 33 and like cc1 can interact with itself. The capability to self-interact makes cc1 and cc2 promising candidates to establish potential crystal contacts between chimeric β3AR-cc molecules. Coiled coils from thermostable proteins are potentially extremely stable and attractive candidates to stabilize β3AR. Towards this aim, the antiparallel coiled-coil domains of seryl-tRNA synthetase (SRS) from Thermus thermophilus (cc3.1) 34 and Pyrolobus fumarii (cc3.2), bacteria that can grow in extreme temperature conditions of up to 85 °C and 122 °C, respectively, were selected. Although high-resolution structures of the isolated thermostable coiled-coils are not available for the proteins, the domain from E. coli has been characterized in detail 35 . Coiledcoil candidates with defined additional α-helical structures instead of the loop connecting the two helices were also selected because they provide a larger surface for crystallization than classical antiparallel coiled coils. cc4 is derived from the pore-forming toxin YaxAB 36 and cc5 represents the microtubule-binding domain from dynein 37 .
Covalent connection of helices by disulfide bonds at their N-or C-terminus is a common approach to stabilize coiled-coil structures 35,38 . Because some of the isolated coiled-coil candidates are potentially not very stable, a second series of β3AR chimeras harboring an intramolecular disulfide bond, termed βAR3-cc_SS, was designed to increase their stability (Supplementary Fig. S1 and Supplementary Table S1).
Based on the secondary structure prediction, truncation of the predicted unstructured N-and C-terminus of human β3AR was carried out. Specifically, 25 N-terminal residues (Pro3 to Thr27) that contain two potential N-glycosylation sites (amino-acid residues Asn8 and Asn26) and the C-terminal 40 residues after the palmitoylation site (Pro369 to Ser408) were deleted.
Residue substitutions were identified that thermostabilized the turkey β1AR 18,39 . The combination mutant was significantly more stable than the native protein when solubilized in dodecylmaltoside (DDM) and in short chain detergents, which allowed its crystallization and structure determination. Because it was shown that the mutations could be transferred from β1AR to β2AR 40 , the β3AR sequence was further modified by introducing the following mutations: Glu36Ala, Met86Val, Ile125Val, Glu126Trp, Tyr234Ala, Phe341Met, and Tyr346Leu 39 .
We selected this structure-based "all-in-one" approach because we never obtained expression of wild-type β3AR. Therefore, we didn't consider truncation of the wild-type a promising approach to obtain expression levels required for structural work. With our in-register coiled-coil fusion approach, we furthermore intend to demonstrate that the testing of multiple fusion sites or multiple constructs at the same fusion site are actually not required.
Only β3AR-cc chimeras fused to thermostable coiled coils exhibit expression. Three different cell lines, HEK293S GnTI − , T-REx-293, T-REx-CHO, were screened for the expression of the 12 β3AR chimeras. Only β3AR-cc3.1 (Fig. 1d) and β3AR-cc3.2 showed reasonable expression levels in T-REx-293 cells. The expression level of β3AR-cc3.1 was higher compared to the one of β3AR-cc3.2. Instead, the other tested variants did not express in any of the cell lines. These results suggest that the stability of GPCRs is an important factor for expression, a hypothesis that is consistent with that we were unable to express wild-type β3AR as a control. They furthermore demonstrate that thermostable coiled coils are promising fusion candidates for GPCR stabilization and that the stabilizing mutations identified for β1AR had no stabilizing effect on β3AR. www.nature.com/scientificreports/ The observation that the disulfide-linked variants of the β3AR-cc3.1 and β3AR-cc3.2 chimeras did not express can be rationally explained by destabilization of the coiled coils upon introducing Cys residues. Hydrophobic amino acids like Leu, Ile or Val at heptad repeat a and d positions are mainly responsible for the stability of a coiled coil and substitution of such a residue usually leads to a significant destabilization of the structure 41,42 .
As a result of its higher expression, we focused on β3AR-cc3.1 and generated stable cell lines expressing chimeric protein. Notably, addition of both tetracycline (2 μg/mL) and sodium butyrate (5 mM) resulted in a three-fold increase in the expression efficiency.
Next, a small-scale purification of β3AR-cc3.1 using StrepTrap Sepharose beads was performed for each detergent as described in the Materials and Methods section. SDS-PAGE analysis demonstrated that β3AR-cc3.1 could be efficiently purified with all the tested detergents. For most detergents, a single band of β3AR-cc3.1 migrating at approximately 42 kDa was detected, but for LMNG, DTG and DDG an additional band of approximately 35 kDa was observed (Fig. 2a). The faster migrating band could represent a SDS-resistant conformation of β3AR-cc3.1 because many membrane proteins migrate faster on SDS-PAGE than their predicted molecular mass 43 .
Therefore, analytical size exclusion chromatography was used in a next step to assess the monodispersity of the purified protein samples. As can be seen in Fig. 2b, a combination of DDM and cholesterol hemisuccinate (CHS) followed by FC12 and HEGA11 were the best detergents in terms of β3AR-cc3.1 monodispersity. Our findings are consistent with the observation that DDM/CHS was previously very successfully used for the purification of several GPCRs 44 . In the following we focused on the three best detergents identified in our experiments.
The thermostable SRS coiled coil significantly stabilizes β3AR. Next, we assessed the thermal stability of β3AR-cc3.1 using the thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) 45 . The assay measures the fluorescence emission of CPM upon forming a covalent bond with the side chain of a free Cys. The free cysteine becomes more readily accessible upon protein thermal denaturation. β3AR-cc3.1 contains 14 cysteine residues. The CPM measurements were carried out in the range of 25-90 °C using a ramping rate of 2 °C/min to warrant equilibrium during unfolding without compromising the integrity www.nature.com/scientificreports/ of CPM. The thermal stability of β3AR-cc3.1 was tested with different ligands (antagonists: SR59230A, L748337 and carvedilol; agonist: carazolol), in Tris-HCl and HEPES buffers, and at different protein concentrations. For β3AR-cc3.1, a melting temperature (T m ) value of 65 °C was obtained in DDM/CHS and carvedilol (Fig. 3). In comparison, the T m of thermostabilized β1AR was 55 °C 18 , demonstrating that our approach is well suited to stabilize GPCRs. As expected, the stability of β3AR-cc3.1 was dependent on the type of detergent that was used. The T m of the purified β3AR-cc3.1 decreased by 8 °C and 10 °C in the presence of HEGA11 and FC12, respectively (Fig. 3).
β3AR-cc3.1 maintains ligand-binding activity. TM helices 5 and 6 play important roles for ligand binding and receptor activation 3 . Because our design approach to link TM helices 5 and 6 by a thermostable antiparallel coiled coil is expected to limit their conformational flexibility, it was important to assess the ligand binding properties of β3AR-cc3.1. To this end, saturation-binding assays were carried out with isolated HEK-239S-GnTI − membranes expressing β3AR-cc3.1.
For these experiments, the antagonist [ 3 H]-dihydroalprenolol (DHA) was used. Although DHA binding to β3AR-cc3.1 was not fully saturated at 300 nM, a K D value of 150 nM was calculated (Fig. 4). A similar K D value of ~ 100 nM was reported for the binding of alprenolol to β3AR 46 , indicating that the engineered GPCR is still capable of binding to the antagonist ligand.
Although the ICL3 of most GPCRs is unstructured, our results are consistent with the observation that extended ICL3 structures are found in some natural GPCRs. For example, the ICL3 of squid rhodopsin 47 and bovine rhodopsin determined from its trigonal crystal 48 both form an extended anti-parallel helical structure  www.nature.com/scientificreports/ that is similar to an antiparallel coiled coil 25 . Although these findings indicate that our engineered GPCR chimera might possibly even still bind G proteins, stabilization of the helix 5-6 interface should directly affect signaling and therefore our chimeras might not be suitable to study GPCR signaling.

The coiled-coil fusion approach is well-suited for the stabilization of other GPCRs. The general
validity of our approach was tested by assessing other GPCRs. In the following, we will focus on the two receptors, the 5-hydroxytryptamine receptor 2C (5HTR2C) and the α-2B adrenergic receptor (α2BAR), that were purified, although successful expression tests have been done for several other GPCRs. Construct design was carried out as described for β3AR-cc3.1. 5HTR2C-cc contains two mutations (Asn204 and Asn205 to Qln), to avoid potential N-glycosylation that could prevent crystallization. No mutations were introduced into α2BAR-cc. After the generation of stable cell lines, both proteins were expressed and purified as described for β3AR-cc3.1 using StrepTag Sepharose affinity chromatography and DDM/CHS as a detergent. 5HTR2C-cc and α2BAR-cc were purified in the presence of the antagonists mianserine and yohimbine, respectively. SDS-PAGE analysis revealed single bands migrating near the 37-kDa marker (Fig. 5). Although these results were very promising for the further characterization of the two chimeric receptors, we focused on β3AR-cc3.1.
β3AR-cc3.1 forms diffracting protein crystals. Because biophysical characterization of β3AR-cc3.1 demonstrated that our coiled-coil-based approach is well suited for quickly and efficiently stabilizing the GPCR, we next aimed at crystallizing the engineered thermostabilized variant bound to carvedilol. Initially, vapor diffusion at 22 °C was employed using a protein concentration of 7.5 mg/mL. Extensive crystallization trials resulted in the formation of 30-70 μm-long needle-like crystals (Fig. 6a) of β3AR-cc3.1 in 0.2 M MgCl 2 , 0.1 M sodium cacodylate pH 6.5 and 50% v/v PEG 200. Typically, the crystals grew within two hours and reached their maximal size after 3-4 days. Crystals displayed diffraction to approximately 22 Å (Fig. 6b). Intensive optimization was performed subsequently using different protein concentrations, additives, pHs, salts, buffers and precipitants, but there was no improvement in diffraction quality. We also tried to crystallize β3AR-cc3.1 using the lipidic cubic phase method, but did not obtain any crystals.

Increasing crystal contacts by replacing the loop connecting the α-helices of the SRS coiled coil by T4L.
Because we were not successful in optimizing conditions to obtain crystals that were suitable for structure determination, we decided to modify β3AR-cc3.1 on the protein level to improve crystal contacts. Truncation or mutation of the antiparallel coiled coil was not considered a valid option because the β3AR-cc3.1_ SS and β3AR-cc3.2_SS variants failed to express, which was probably due to destabilization of the proteins. Although it has been reported that the loop connecting the two helices of an antiparallel coiled coil is crucial for the stability of the structure, it was demonstrated that substitution of the loop by a disulfide bond flanking the heptad repeats restored coiled-coil formation 35 . Although substitution of the connecting loop might alter the thermal stability of the antiparallel coiled coil, we decided to replace it with phage T4L. We selected T4L because it has been very successfully used as a fusion protein to crystallize and determine the structures of several GPCRs 11,19,20 . Because it crystallizes easily under many different conditions, T4L is considered an ideal fusion partner to establish crystal contacts. Another criterium for of choice was the existence of a β2AR crystal structure in which ICL3 was replaced by T4L. For the design, we used the structure-guided strategy described for the construction of β3AR-cc3.1. More specifically, we grafted the experimentally determined boundary of T4L to the coiled-coil like structure seen in β2AR on the antiparallel coiled coil of β3AR-cc3.1 (Fig. 7a,b). www.nature.com/scientificreports/ Expression of the resulting variant, termed β3AR-cc-T4L, was only observed in T-REx-CHO cells (Fig. 7c). Despite we were not able to adapt stable cells for growth in suspension, we managed to purify a small amount of β3AR-cc-T4L for characterization from adherent T-REx-CHO cells using the best condition described for β3AR-cc3.1 (detergent combination DDM/CHS and antagonist carvedilol). Notably, β3AR-cc-T4L was significantly more stable than β3AR-cc3.1 and yielded a 10 °C higher T m value of 75 °C (Fig. 7d). Therefore, β3AR-cc-T4L represents a promising candidate for crystallization.  www.nature.com/scientificreports/ Although the structure of the β3AR in its active state has recently been reported 49 , β3AR-cc-T4L is also a potential candidate for structure determination of the inactive receptor by cryo-EM. It has a similar molecular weight as organic cation transporter-3 (62 kDa) for which a resolution of 3.2 Å has been obtained in recent studies 50 . Furthermore, to aid crystallization, β3AR-cc-T4L was designed to contain no flexible regions, which should also benefit cryo-EM studies. Therefore, we next tested the suitability of the engineered GPCR for cryo-EM analysis. Inspection of β3AR-cc-T4L molecules by transmission electron microscopy (TEM) after negative staining yielded a homogeneous distribution of uniformly appearing compact particles (Fig. 7e). This finding demonstrates that structure determination β3AR-cc-T4L by cryo-EM might be a promising approach, in particular in combination with a universal DARPin or nanobody that binds to T4L.

Conclusions
Structure determination of inactive-state GPCRs still mostly relies on X-ray crystallography. Based on the available GPCR crystal structures, a combination of protein stabilization and accessible soluble surface to establish crystal contacts would represent an ideal tool for crystallization. Towards this aim, we identified thermostable antiparallel coiled coils a such a tool. Preplacement of ICL3 of β3AR by cc3.1 resulted in significant stabilization of the GPCR while retaining its ligand-binding properties. Furthermore, we were also able to stabilize two other GPCRs, 5HTR2C and α2BAR (Fig. 5), demonstrating that this approach is generally suitable for the stabilization of GPCRs. Stabilization provides an attractive means for GPCRs that have only limited stability or are difficult to express. Furthermore, stabilized GPCRs appear also be of considerable interest for drug discovery applications because many drugs target the inactive state of the receptors.
Although we managed to crystallize the β3AR/coiled-coil chimera, the quality of the crystals even after extensive optimization was not suitable for structure determination. To supply additional surface for promoting crystal contacts, we replaced in a structure-based approach the loop connecting the helices of the antiparallel coiled coil by T4L. Although expression levels are currently not suitable for crystallization, we were able to show that the engineered GPCR is even more stable than the β3AR/coiled-coil chimera. TEM experiments demonstrated that GPCR-ccT4L fusions also represent promising candidate molecules for structure elucidation by cryo-EM.
The generation of the engineered GPCRs that led to a crystal structure often required many constructs for the optimal insertion of fusion proteins. It is therefore noteworthy that our structure-based approach is simple and straightforward. For all the examples described in this study, only the design of one construct was necessary to produce each protein.

Materials and methods
Constructs. Synthetic genes encoding the human β3 adrenergic receptor (β3AR, amino-acid residues Ala2 to Leu368 lacking Pro3 to Thr27) with the antiparallel coiled-coil sequences described in this study (Supplementary Table S1) inserted between Ala231 and Glu286 were codon-optimized for expression in human cells (Genewiz). Synthetic genes codon optimized for human cell expression and spanning amino-acid residues His3 to Trp450 of the human α-2B adrenergic receptor (α2BAR) and amino-acid residues Gly43 to Pro395 of the rat 5-hydroxytryptamine receptor 2C (5HTR2C) with cc3.1 inserted between Ala199 and Glu366 and Leu242 and Glu308, respectively, were purchased from Genewiz. The β3AR sequences also include the potentially thermostabilizing mutations Glu36Ala, Met86Val, Ile125Val, Glu126Trp, Tyr234Ala, Phe341Met, and Tyr346Leu 18,39 . In 5HTR2C two potential N-glycosylation sites (Asn204 and Asn205) were mutated to Gln. Insert sequences were further modified to contain a hemagglutinin signal sequence followed by a modified FLAG tag at the N-terminus and a TwinStrep tag that can be removed by thrombin or HRV 3C cleavage at the C-terminus ( Supplementary  Fig. S1). The full-length wild-type human β3AR cDNA sequence was used as a control. Insert sequences were subcloned into mammalian expression vectors pACMV-tetO 51 and pcDNA4/TO (ThermoFisher Scientific).
Stable cell lines were generated by using geneticin (G-418, 0.2 mg/mL) or zeocin (0.4 mg/mL) for pACMV-tetO and pcDNA4/TO, respectively. Individual colonies (24 for each receptor construct) typically appeared after 14 days and were isolated and expanded as described before 51 . Protein expression was induced with tetracycline (2 μg/mL) and sodium butyrate (5 mM) and cells further incubated for 72 h. For each clone, the expression level of the recombinant protein was assessed by western blotting, and the best clones were selected for further largescale expression in suspension. Stably transfected cells were grown in suspension in a final volume of 5 × 1 L of PEM media (5% fetal calf serum (FCS), 1% (pyrrolidone carboxylic acid) PSA, 2.5 mM Glutamax). Expression was induced upon reaching a cell density of ~ 3 × 10 6 cells/mL as described above. Cells were harvested after 72 h by centrifugation at 2500g, frozen in liquid nitrogen and stored at − 80 °C for protein purification or washed twice with PBS prior to freezing for membrane preparations.
Transmission electron microscopy (TEM). Negative stain images of B3ARccT4L were made by applying one drop of protein sample (10 µg/mL) onto a glow-discharged carbon coated copper EM grid for 60 s. The grid was washed with three drops of distilled water, one drop of 2% uranyl acetate and subsequently placed on a drop of 2% uranyl acetate for 10 s, blotted with a filter paper and allowed to air dry. Negatively stained EM grids were observed on a Talos L120C microscope (Thermo Fisher Scientific, USA), operated at 120 kV. Images were recorded at a nominal magnification of 120,000 × using a Ceta CMOS camera, corresponding to a pixel size of 1.2 Å/pixel on the specimen. TEM images were analyzed by the image analysis tool ImageJ (NIH).

Data availability
All the data used in this paper are available from the corresponding author on reasonable request.